Pathologies of sexual development are common in humans, reflecting the precarious processes of sex determination and sexual differentiation. The gonad forms as a bipotential organ, composed of cells that can differentiate as testis or ovarian cell types. The primary sex-determining step is the fate decision that occurs in the somatic supporting cell lineage. We previously showed that supporting cells are initially balanced between Sertoli (testis) and follicle (ovary) fates by opposing and antagonistic signaling networks. In XY embryos, this balance is disrupted by the transient expression of the Y-linked gene Sry in supporting cell precursors. Our global analysis of the gonad transcriptome revealed two concurrent networks in the XY bipotential gonad, one male and one female. This data suggests that male sex determination requires both the activation of male pathway genes and the stable repression of the underlying female pathway during male fate determination. This idea is supported by the finding that loss of function of the polycomb protein M33/CBX2 in mice or humans results in male to female sex reversal. During this funding period, we found that Notch signals in rapidly dividing cells that give rise to the supporting cell precursors, whereas NUMB localizes basolaterally in these cells, but is symmetrically distributed in cells in the interior of the gonad that establish expression of either SRY and its downstream target SOX9, or the earliest marker of female supporting cell precursors, FOXL2. We found that NUMB-expressing supporting cell precursors of both sexes are p27/p21-positive and arrested in G0/G1 of the cell cycle at the stage when differentiation begins. We hypothesize that Numb mediates cell cycle arrest, which is required for chromatin remodeling through the activity of the polycomb complex (PRC1) to establish and/or maintain Sertoli and granulosa cell fate. In this proposal, we will determine how gonadal cells approach and execute the sex-determining cell fate decision through the following aims: (1) Determine the role of Notch/Numb signaling and cell cycle arrest in specification of the supporting cell lineage;(2) Determine whether M33/PRC1 is required to establish the supporting cell lineage and to maintain Sertoli or granulosa cell fate. Integration of the cell cycle with cell fate specification and epigenetic reprogramming will have broad relevance to the fields of organogenesis and stem cell biology and will provide insight into the developmental basis for disorders of sexual development and infertility. PUBLIC HEALTH RELEVANCE: Relevance Statement This proposal will fill a significant gap in our understanding of how the cell cycle is integrated with cell fate specification and epigenetic reprogramming. Results will have broad relevance to the fields of organogenesis, reprogramming, and stem cell biology. Within the field of reproductive biology, results will provide insight into the epigenetic basis for disorders of sexual development and infertility such as those associated with mutations in ATRX, M33/CBX2, and the GATA4/FOG2 complex.